致羔羊腹泻奇异变形杆菌的分离鉴定及其ompA基因重组乳酸乳球菌口服疫苗研制

发布时间：2018-11-10 20:19

【摘要】：2015年春,山东滕州某规模化羊场新生羔羊出现严重的腹泻,并且部分羔羊死亡。对该羊场病死羔羊进行剖检后,共分离到四株细菌(分别命名为PM1、PM2、PM3、PM4)。对所分离的细菌进行形态特征、生化试验以及16S rDNA遗传分析,判定该四株菌均为奇异变形杆菌。16S rDNA系统发育及同源性分析显示:四株分离菌与其它11株(NCBI收录)奇异变形杆菌的同源性为99%以上,其中与中国Hμ株、马来西亚PPB3株以及印度BAB-199株位于一个分支上,且与中国的Hμ株的进化关系最近;四株分离菌之间的同源性为100%,判断为同一株菌。细菌生长曲线、药敏试验、毒力试验、毒素测定试验以及游散行为分析结果表明,该分离菌于14 h左右达到生长稳定期,且对环丙沙星、左氟沙星最为敏感;对小白鼠有强致病性,分离菌培养液的无菌滤液对小白鼠无毒性作用;在含0.5%琼脂的LB平板上迁徙速度最快,且在含琼脂1.5%和2.0%的LB平板上呈圆环样周期运动。该研究结果为羊奇异变形杆菌病的防控提供了基础。本试验构建了含羊奇异变形杆菌外膜蛋白A(ompA)编码基因的重组载体pNZ8149-ompA,并将其电转入乳酸乳球菌NZ3900中。利用Westen blot检测目的蛋白的表达,间接免疫荧光试验及流式细胞仪检测目的蛋白在乳酸乳球菌中的定位。将重组乳酸乳球菌灌胃BALB/c小鼠,分别于口服后的1、2、3、4、5、6、7 d之后取其十二指肠、空肠、回肠、盲肠的肠道冲洗液,通过平板菌落计数法检测肠道内的乳酸乳球菌定植情况。Western blot检测到目的蛋白于49kDa处出现特异性条带;间接免疫荧光试验检测到重组乳酸乳球菌菌体表面出现绿色荧光;流式细胞仪检测到重组乳酸乳球菌表面荧光强度明显强于空质粒对照组。重组乳酸乳球菌灌服小鼠后能在肠道黏膜的不同部位以一定比例存活,并于口服后6 d达到定植高峰,7 d后在十二指肠、空肠、回肠和盲肠定植率分别占第1 d的15.23%、24.19%、48.57%、40.07%。本试验表明羊奇异变形杆菌ompA能够稳定表达于乳酸乳球菌表面,且重组菌在小鼠肠道内具有良好的定植能力,定植规律为回肠盲肠空肠十二指肠。这为研究乳酸乳球菌作为羊奇异变形杆菌口服疫苗及其对小鼠肠道免疫机理的影响提供试验基础。以泰山松花粉多糖(TPPPS)作为口服疫苗佐剂,探究其对小鼠免疫功能作用的影响,结果表明,单纯的重组乳酸乳球菌口服疫苗能诱导特异性IgA和IgG、IL-2、IL-4和IFN-γ的产生,促进T淋巴细胞增殖,且免疫后小鼠可显著抵抗奇异变形杆菌于肠道内定植。此外,与单纯重组乳酸乳球菌口服疫苗相比,TPPPS佐剂可诱导更高水平的IgA、IgG、IL-2、IL-4和IFN-γ,以及T淋巴细胞增殖反应。综上所述,乳酸乳球菌联合TPPPS佐剂制备口服疫苗,提供了一种预防奇异变形杆菌感染的新型方法。
[Abstract]:In the spring of 2015, severe diarrhea and the death of some lambs occurred in a large scale sheep farm in Tengzhou, Shandong Province. Four strains of bacteria (named PM1,PM2,PM3,PM4) were isolated from the dead lamb. Morphological characteristics, biochemical tests and 16s rDNA genetic analysis were performed on the isolated bacteria. The phylogeny and homology analysis of 16s rDNA showed that the homology of the four isolates with other 11 strains (NCBI included) was more than 99%, and the homology was more than 99% with that of H 渭 strain in China. The Malaysian PPB3 strain and the Indian BAB-199 strain are located in one branch and have the closest evolutionary relationship with H 渭 strain in China. The homology of the four isolates was 100, which was determined to be the same strain. The results of bacterial growth curve, drug sensitivity test, virulence test, toxin test and swimming behavior analysis showed that the isolated bacteria reached the stable growth stage at about 14 h, and levofloxacin was the most sensitive to ciprofloxacin. It has strong pathogenicity to mice, and the aseptic filtrate of isolated bacteria culture medium has no toxic effect on mice. The migration rate was the fastest on the LB plate containing 0.5% Agar and the circular cycle motion was observed on the LB plate containing 1.5% Agar and 2.0% Agar. The results provide a basis for the prevention and control of Proteus mirabilis disease in sheep. In this study, the recombinant vector pNZ8149-ompA, containing A (ompA) encoding gene of Proteus mirabilis outer membrane protein was constructed and electrotransferred into NZ3900 of Lactococcus lactis. The expression of target protein was detected by Westen blot, the localization of target protein in Lactococcus lactis was detected by indirect immunofluorescence assay and flow cytometry. BALB/c mice were perfused with recombinant Lactococcus lactis. After oral administration of Lactococcus lactis, the intestinal irrigation fluid of duodenum, jejunum, ileum and cecum were collected after oral administration for 7 days. The colonization of Lactococcus lactis in intestinal tract was detected by plate colony counting method. The specific band of target protein was found in 49kDa by. Western blot. Indirect immunofluorescence assay detected green fluorescence on the surface of recombinant Lactococcus lactis, and flow cytometry showed that the fluorescence intensity of recombinant Lactococcus lactis was significantly stronger than that of blank plasmid control group. Recombinant Lactococcus lactis could survive in different parts of intestinal mucosa in certain proportion after oral administration, and reached the colonization peak at 6 days after oral administration, and 15.23 percent in duodenum, jejunum, ileum and cecum at 7 days after oral administration. 24.19 there are 48.57 and 40.07. The results showed that Proteus mirabilis ompA could be stably expressed on the surface of Lactococcus lactis, and the recombinant bacteria had good colonization ability in mouse intestine, and the colonization rule was jejunum duodenum of ileum caecum. This provides the experimental basis for the study of Lactococcus lactis as an oral vaccine of Proteus mirabilis and its effect on the intestinal immune mechanism of mice. The effects of Taishanpine Pollen Polysaccharide (TPPPS) as adjuvant on immune function in mice were investigated. The results showed that the recombinant Lactococcus lactis oral vaccine could induce specific IgA and IgG,IL-2,. The production of IL-4 and IFN- 纬 promoted the proliferation of T lymphocytes, and the immunized mice could significantly resist the colonization of Proteus mirabilis in the intestine. In addition, TPPPS adjuvant could induce higher levels of IgA,IgG,IL-2,IL-4 and IFN- 纬, as well as T lymphocyte proliferation, compared with recombinant lactococcus lactis oral vaccine. To sum up, Lactococcus lactis combined with TPPPS adjuvant to prepare oral vaccine provides a new method to prevent Proteus mirabilis infection.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.26

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